The present invention relates to a method for assaying human pepsinogen I or human pepsin I in the human body fluid (such as gastric juice, blood, urine etc.) as diagnostic marker of gastric diseases such as gastric cancer, gastric ulcer etc., and a peptide used as substrate in such a method.
More detailed, the present invention relates to
(1) a peptide of the formula (I)
R-X-ProIleGlu-W-Y-Zxe2x80x83xe2x80x83(I)
(wherein R is hydrogen, an amino-protective group, or a residue containing one or two D-amino acid or L-amino acid,
X is Lys or Arg residue,
W is Phe or Phe(NO2) residue (in which Phe(NO2) is p-nitrophenylalanine residue.),
Y is Phe, Phe(NO2), Tyr, Dit, Nle, Leu, Asp(OBzl) or Met residue (in which Dit is 3,5-diiodotyrosine residue, Nle is norleucine residue, OBzl is benzyloxy and Phe(NO2) is as defined hereinbefore.), and
Z is an aniline derivative residue, an aminocoumarine derivative residue or an aminonaphthalene derivative residue.),
or an acid addition salt thereof and,
(2) a method for assaying human pepsinogen I or human pepsin I which is characterized by digesting a peptide of the said formula (I) or an acid addition salt thereof by human pepsin I which is obtained by activation of human pepsinogen I in a sample or human pepsin I in a sample to obtain an amino acid derivative of the formula (II)
H-Y-Zxe2x80x83xe2x80x83(II)
(wherein all the symbols are as defined.),
digesting the obtained amino acid derivative by aminopeptidase to obtain an aniline, aminocoumarine or aminonaphthalene derivative of the formula Z-H and then
detecting the obtained aniline, aminocoumarine or aminonaphthalene derivative.
It is known that the pepsinogen secretion is parallel to gastric acid secretion and that human serum or urine pepsinogen levels is also parallel to gastric pepsinogen secretion. The above pepsinogen exists as pepsinogen in the body fluid such as blood or urine except for gastric juice, on the other hand, it exists as pepsin in gastric juice. It is said that the blood or urine pepsinogen I level of the patient with atrophic gastritis decreases and that human blood pepsinogen I and pepsinogen II levels increase in case of gastric ulcer. In addition, the level of pepsinogen I is said to decrease in the patient with gastric cancer (Japanese Patent Application Kokai Hei 7-304800). Therefor, a assaying the level of human pepsinogen I in human blood or urine may be useful for diagnosis of gastric diseases such as gastric cancer, gastric ulcer etc. in early stage.
As for a method for assaying human pepsin which was obtained by activation of human pepsinogen, a method using human serum protein etc. in urine and serum based on its digesting activity has been known (Clin. Chem., 15, 1, 42-55 (1969)). The significance of clinical trial using such a method has been discussed, but it requires a long time. In addition, its accuracy was not good, so such a method has been of no practical use. Further, the results means the activity to digest protein, so it was reflected on the total activities of both pepsin I and pepsin II. Therefore it is impossible to determine the human serum pepsinogen I specifically.
Recently, a method for assaying human pepsinogen in urine (uropepsin) indirectly, based on inactivation of an aciduric enzyme by activated pepsin was proposed (Japanese Patent Application Kokai Hei 7-155198). But the substrate used in this method did not show the specificity for pepsin I. It is said that the pepsinogen in urine is pepsinogen I. But, pepsin II may be also secreted in urine in some body condition, so it is difficult to determine the accurate level of pepsin I. It is impossible to assay the level of pepsinogen I in human serum specifically.
As for a method for assaying pepsinogen I, radio immunoassay (Gastroenterology, 66, 494 (1974)) and enzyme immunoassay (Japanese Patent Application Kokai Hei 7-304800) using a specific anti-body have been practical use, but these methods cause a radioactive pollution and require a long time and complicated procedure for determination.
As for a method for assay using synthesized substrate, for example, it was described that a peptide of the formula (A-1)
LysProIleGluPhePhe(NO2)ArgLeuxe2x80x83xe2x80x83(A-1)
(wherein Phe(NO2) is as defined hereinbefore.) was used as substrate in assaying inhibitory effect of some compounds on human pepsin I in the paper of J. Med., Chem., 36, 2614 (1993). That is to say, a peptide of the formula (A-1) was digested by human pepsin I to obtain a peptide of the formula (A-2)
Phe(NO2)ArgLeuxe2x80x83xe2x80x83(A-2)
(wherein Phe(NO2) is as defined hereinbefore,)
and the obtained peptide of the formula (A-2) was used in the assaying inhibitory activity on enzyme based on decrease of absorbance at 234xcx9c324 nm as an index.
But this paper did not disclose that such a peptide of the formula (A-1) may be as a substrate for human pepsin I. Therefore, it is uncertain whether this peptide has a specificity for human pepsin I, or not. In addition, the chromophore of this peptide is Phe(NO2), so it is expected that the accuracy of the method using this peptide is one tenth or less to compare with p-nitroaniline (abbreviated as pNA). Further, it is impossible to be used in automated clinical analyzer due to detection at 234xcx9c324 nm.
In CS-261172, a peptide of the formula (B-1)
X-A-B-Phe-D-pNAxe2x80x83xe2x80x83(B-1)
(wherein X is hydrogen, C3xcx9c5 carboxylalkylcarbonyl or C1xcx9c5 alkylcarbonyl,
A is pyroglutamic acid (abbreviated as pGlu), Asp, Glu or Gly residue or 2-oxoimidazoline-1-yl-carbonyl,
B is His, Gly or Pro residue,
D is Phe, Leu, Nle, Met or S-C1xcx9c3 alkyl-Cys residue, and pNA is p-nitroaniline residue.)
was used as a substrate in assaying activities of pepsin I, pepsin II and chymosin.
But, there was neither description nor suggestion about the specificity of this peptide for human pepsin I.
In the example, a peptide of the formula (B-2)
pGluHisPhePhe-pNAxe2x80x83xe2x80x83(B-2)
(wherein pGlu and pNA are as defined hereinbefore.)
was used in assaying activity of pig pepsin. This pig pepsin was not purified and thought to be an mixture of pepsin I and pepsin II. So, it is not expected that this substrate possesses a specificity for human pepsin I.
There is a common part in chemical structure between these substrates described in the above two references (J. Med. Chem., 36, 2614 (1993) and CS-261172) and the substrate of the present invention, but these compounds are distinct from this substrate in total chemical structure. There are neither description nor suggestion that the peptides of the present invention have a specificity for human pepsin I in these references.
In addition, in the paper of Anal. Biochem., 234, 113 (1996), a peptide of the formula (C-1)
Abz-AlaAlaPhePheAlaAla-Dedxe2x80x83xe2x80x83(C-1)
(wherein Abz is o-aminobenzoyl and Ded is N-2,4-dinitrophenylethylenediamine.),
or a peptide of the formula (C-2)
Abz-AlaAlaPhePheAlaAla-pNAxe2x80x83xe2x80x83(C-2)
(wherein Abz and pNA are as defined hereinbefore.)
were used as substrate in assaying activities of human pepsin I, human pepsin II, human cathepsin D and HIV protease by fluorophotometry.
In this paper, the fluorescent changes caused by modification of peptide was discussed. Particularly, a peptide of the formula (C-1 ) seemed to be suggested to have specificity for human pepsin I from the experimental result. But, the level of specificity is insufficient. A peptide of the formula (C-1) was distinct from a peptide of the present invention in structure. Therefore, it is not expected that the peptide of the present invention has the specificity for human pepsin I from the peptide of the reference.
Further, in the paper of Proc. Soc. Exp. Biol. Med., 122, 700 (1966), a peptide of the formula (D-1)
Ac-Phe-Ditxe2x80x83xe2x80x83(D-1)
(wherein Ac is acetyl and Dit is 3,5-diiodotyrosine residue.)
was used as a substrate in specifically assaying human pepsin I. But this method required a long time and complicated procedure. This method is of no practical use due to the above reason and low accuracy and low sensitivity.
A peptide of the formula (D-1) was distinct from a peptide of the present invention in structure. Therefore, it is not expected that the peptide of the present invention has the specificity for human pepsin I from the peptide of the reference.
The present inventors have been studying to dissolve these problems of the related arts and to find a substrate which is high-sensitive (being high rate of enzyme reaction i.e., digesting a substrate by human pepsin I at a high rate and/or being able to produce efficient coloring) and specific for human pepsin I, and then have succeeded in synthesizing a substrate (peptide) which is sensitive and specific for human pepsin I. By using this substrate, it become to possible to determine pepsin I for short time to compare with the method of related arts and to determine pepsinogen I using automated clinical analyzer.
That is to say, the present invention relates to
1) a peptide of the formula (I)
R-X-ProIleGlu-W-Y-Zxe2x80x83xe2x80x83(I)
(wherein R is hydrogen, an amino-protective group, or a residue containing one or two D-amino acid or L-amino acid,
X is Lys or Arg residue,
W is Phe or Phe(NO2) residue (in which Phe(NO2) is p-nitrophenylalanine residue.),
Y is Phe, Phe(NO2), Tyr, Dit, Nie, Leu, Asp(OBzl) or Met residue (in which Dit is 3,5-diiodotyrosine residue, Nle is norleucine residue, OBzl is benzyloxy and Phe(NO2) is as defined hereinbefore.), and
Z is an aniline derivative residue, an aminocoumarine derivative residue or an aminonaphthalene derivative residue.),
or an acid addition salt thereof,
2) a peptide described in the above 1), wherein Y is Phe, Phe(NO2), Tyr or Nle residue wherein all the symbols are as defined in the above 1),
3) a peptide described in the above 1) or 2), wherein R is hydrogen or C1xcx9c6 alkylcarbonyl,
4) a peptide of the formula (Ia)
Nxcex1-Ac-LysProIleGluPheNle-pNAxe2x80x83xe2x80x83(Ia)
(wherein Ac is acetyl, Nle is norleucine residue, pNA is p-nitroaniline residue.),
a peptide of the formula (Ib)
LysProIleGluPheNle-pNAxe2x80x83xe2x80x83(Ib)
(wherein all the symbols are as defined hereinbefore.),
a peptide of the formula (Ic)
LysProIleGluPheTyr-pNAxe2x80x83xe2x80x83(Ic)
(wherein all the symbols are as defined hereinbefore.),
a peptide of the formula (Id)
LysProIleGluPhePhe-pNAxe2x80x83xe2x80x83(Id)
(wherein all the symbols are as defined hereinbefore.),
a peptide of the formula (Ie)
LysProIleGluPhePhe(NO2)pNAxe2x80x83xe2x80x83(Ie)
(wherein Phe(NO2) is p-nitrophenylalanine residue, and the other symbol is as defined hereinbefore.),
or a peptide of peptide of the formula (If)
Nxcex1-AcLysProIleGluPhe(NO2)-NlepNAxe2x80x83xe2x80x83(If)
(wherein all the symbols are as defined hereinbefore.), or acid addition salts thereof,
5) a method for assaying human pepsinogen I or human pepsin I characterized by
digesting a peptide of the formula (I) described in the above 1)
(wherein all the symbols are as defined in the above 1).), or an acid addition salt thereof, by human pepsin I which is obtained by activation of human pepsinogen I in a sample or human pepsin I in a sample to obtain an amino acid derivative of the formula (II)
H-Y-Zxe2x80x83xe2x80x83(II)
(wherein all the symbols are as defined in the above 1).),
digesting the obtained amino acid derivative by aminopeptidase to obtain an aniline, aminocoumarine or aminonaphthalene derivative of the formula Z-H and then detecting the obtained aniline, aminocoumarine or aminonaphthalene derivative,
6) a method for assaying described in the above 5) using a peptide of the formula (Ia), (Ib), (Ic), (Id), (Ie) or (If) wherein all the symbols are as defined in the above 4) or an acid addition salt thereof,
7) a kit for assaying human pepsinogen I or human pepsin I which is characterized by comprising a peptide of the formula (I) (wherein all the symbols are as defined in the above 1).), or an acid addition salt thereof as a substrate and aminopeptidase.